In vitro site-directed mutagenesis, which involves the substitution of single amino acids in a protein by changing the relevant base residues in the encoding DNA, has proved to be a powerful method in protein engineering. This technique typically requires information on the structure-function relationship of the protein under study in order to provide a rationale for generating mutants with altered properties. In contrast, random mutagenesis of the DNA region of interest coupled with adequate screening or selection procedures provides an alternative and general method for the generation of DNA, RNA or protein species with improved or novel functions in the absence of initial structural information.
Several methods for the generation of mutants of large DNA fragments have been described and involve using pools of random sequence synthetic oligonucleotides (Matteucci & Heyneker, Nucl. Acids Res. 1983 11, 3113; Wells et al., Gene 1985 34, 315; Nerr et al., DNA 1988 7, 127 and references therein), chemical modification of the target sequence (Kadonaga & Knowles, Nucl. Acids Res. 1985 13, 1733; Meyers et al, Science 1985 229, 242, and references described therein); or base misincorporation using an error-prone polymerase (Lehtovaara et al, Protein Eng. 1988 2, 63).
The synthetic oligonucleotide approach is restricted by the length of the DNA amenable to chemical synthesis, whilst the chemical approach is often labour intensive. In other approaches, random mutations are generated using the polymerase chain reaction (PCR). One such method relies exclusively on the intrinsic error frequency of Taq DNA polymerase, resulting in about 0.5.times.10.sup.-3 mutations per base pair (Zhou, Nucl. Acids Res. 1991 19, 6052). In an improved variation of this method the target sequence of interest is copied under conditions which further reduce the fidelity of DNA synthesis catalysed by Taq DNA polymerase e.g. by the addition of the cofactor manganese and by the use of high concentrations of magnesium and the relevant deoxynucleoside triphosphates (dNTPs--see Leung et al., Techniques 1989 1, 11). Using the latter procedure mutation frequencies in the order of 20.times.10.sup.-3 mutations per base pair have been claimed.
An alternative approach to PCR-based random mutagenesis is to replace, partially or fully, the 5'-triphosphates of the four natural nucleosides by the triphosphates of nucleoside analogues which display ambivalent base pairing potential. To our knowledge this approach has only been attempted using deoxyinosine triphosphate--dITP (Spee et al, Nucl. Acids Res. 1993 21, 777; Ikeda et al, J. Biol. Chem. 1992 267, 6291). However, this analogue is a poor substrate for Taq Polymerase and cannot support DNA synthesis when replacing any of the four normal dNTPs. As a result, four separate PCR reactions are required containing dITP and three dNTPs in equal concentrations together with limiting concentrations of the fourth dNTP. The four separate PCR products are then pooled and cloned (Spee et al., cited above).
A general feature of the above procedures is that the yield of mutant sequences is low and that the pattern of mutations is heavily biased towards transitions (pyrimidine-pyrimidine or purine-purine substitutions). In addition, with the last two methods, undesirable base additions or deletions occur at an appreciable rate.
In an alternative approach, it was envisaged that the 5'-triphosphates of a pyrinidine or purine nucleoside analogue capable of inducing transition mutations in combination with other triphosphate analogues capable of causing transversion mutations would allow efficient random mutagenesis via PCR. The nucleoside analogues P (Kong Thoo Lin & Brown, Nucl. Acids Res. 1989 17, 10373) and K (Brown & Kong Thoo Lin, Carbohydrate Res. 1991 216, 129), (structures 1 and 3 respectively, shown in FIG. 1) have previously been incorporated into oligonucleotides and demonstrate ambivalent base pairing potential, as illustrated for P in FIG. 2. That is, P forms base pairs of equivalent stability with adenine and guanine. Likewise K forms base pairs with closely similar stabilities with thymine and cytosine. In addition, template DNA containing these analogues is recognised by polymerases such as Taq polymerase in PCR and Sequenase.TM. in DNA sequencing (Kong Thoo Lin & Brown, Nucleic Acids Res. 1992 20, 5149; Kamiya et al., Nucleosides & Nucleotides 1994 13, 1384; Brown & Kong Thoo Lin, Collect. Czech. Chem. Commun. (Special issue), 1990 55, 213). The present inventors considered that other analogues, e.g. 2'-deoxy-8-hydroxyguanosine 5'-triphosphate, abbreviated as 8oxodGTP (Pavlov et al, Biochemistry 1994 33, 4695), shown as structure 5 in FIG. 1, might be valuable in this context in order to generate transversion mutations.